Laboratory of Developmental Biology, Institute of Animal Physiology and Genetics Academy of Science of Czech Republic, v.v.i., Libechov, Czech Republic.
Department of Veterinary Sciences, Czech University of Life Sciences in Prague, Prague, Czech Republic.
Biol Reprod. 2019 Apr 1;100(4):896-906. doi: 10.1093/biolre/ioy254.
The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.
在胚胎植入前发育过程中母体蛋白降解的机制尚未阐明。人们认为大量的母体蛋白通过泛素-蛋白酶体系统降解。在这项研究中，我们专注于 SCF（Skp1-Cullin-F-box）复合物在早期牛胚胎发生中的作用。我们使用 MLN4924（一种由 neddylation 控制的 SCF 复合物连接酶抑制剂）来抑制它们。在 MLN4924 中成熟的卵母细胞可以受精，但我们发现体外受精后没有卵丘细胞扩张和多精受精。我们还发现 MLN4924 处理后胚胎发育明显恶化。在用 MLN4924 从四细胞到晚期八细胞阶段处理后，我们发现它们的发育有明显的延迟；然而，一些处理过的胚胎后来能够到达囊胚阶段。我们发现 EGA 标记物 PAPOLA 和 U2AF1A 的 mRNA 水平降低，这可能与这种发育延迟有关。用 MLN4924 培养会导致 MLN4924 处理的卵母细胞和胚胎中的蛋白质水平显著增加；在卵丘细胞中没有发现这种变化。为了检测受 MLN4924 处理影响的蛋白质，我们对选定的蛋白质（SMAD4、核糖体蛋白 S6、着丝粒蛋白 E、P27、NFKB 抑制剂 alpha、RNA 结合基序蛋白 19）进行了 Western blot 分析。在处理过的胚胎或卵母细胞中均未检测到蛋白质水平的统计学显著增加。总之，我们的研究表明，SCF 连接酶对于卵母细胞的正确成熟、卵丘细胞的扩张、受精以及牛早期胚胎植入前的发育是必要的。
